Cellular interactions between antigen (Ag)-specific T cells and Ag-presenting cells (APCs) can trigger either productive or aborted immune responses. The cellular outcomes depend on the state of the T cells, their previous exposure to Ag, and the APCs. This application addresses the central hypothesis that the molecular architecture of the membrane of T cells plays a decisive role in influencing Ag-sensitivity and cellular outcomes. Aim 1 will use immuno-electron microscopy, FRET and Quantum Dot blinking to test the hypothesis that antigenic stimulation of naTve T cells results in long-term formation of TCR micro-clusters that facilitate responses to recall Ags. Aim 2 will use multi-dimensional digital imaging and biochemical analysis to identify specific antigen-induced spatial-temporal molecular alterations in Immunological Synapses that trigger altered immune responses. This Aim will test the hypothesis that disproportionate activation of Fyn and Lck in the Immunological Synapse can cause unresponsiveness and would address the functional significance of the Immunological Synapse. Aim 3 will test more directly the roles of selective activation of Fyn or Lck on the structure of the synapse. This Aim will use novel kinase-null T cellls and switchable mutated inactive tyrosine kinases Fyn and Lck that can be chemically rescued rapidly and reversibly (within 5 min) in T cells. Uncovering the mechanisms by which T cells can sense Antigen and how this recognition determines the cellular response is the long-term objective of all 5 projects in this Program. There is an extremely high synergy with all other projects. Aim 1 synergizes with Project 1 that will study TCR clustering in the membrane of CDS cells. Aim 2 synergizes with Projects 3, 4 and 5, which will use the specialized imaging tools in their own structure function studies of Sproutyl, TFII-I and CARD11 and their associations with the Immunological Synapse. Aim 3 would synergize with Project 4 where all the tools for chemical rescue of Fyn and Lck will be developed. These studies that combine uniquely the use of several highly novel experimental approaches are very likely to generate significant better understanding of the integrated events that regulate immune responses to infectious pathogens. This knowledge may lead to the design of more effective vaccines, better immune surveillance protocols, and new immunosupressive drugs.